South African Journal of Botany 130 (2020) 45
53

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Evaluation of the antioxidant and anti-arthritic potential of Zingiber

officinale Rosc. by in vitro and in silico analysis
Selvakumar Murugesan, Meenakshi R. Venkateswaran, Sasidharan Jayabal,
Sureshkumar Periyasamy*
Department of Biotechnology, Anna University, BIT-Campus, Tiruchirappalli 620024, Tamil Nadu, India

A R T I C L E I N F O A B S T R A C T

Article History: Rheumatoid Arthritis (RA) is an autoimmune disorder that leads to joint and bone destruction. The available
Received 30 July 2019 anti-inflammatory chemo drugs are associated with adverse side-effects providing only temporary relief.
Revised 6 November 2019 Herbal medicine can act as an alternative source for RA treatment with improved efficacy, lesser toxicity and
Accepted 11 December 2019
side effects. An attempt was made to evaluate the antioxidant and anti-arthritic potential of ginger (Zingiber
Available online xxx
officinale Rosc.). Crude ginger extracts were prepared with different organic solvents using soxhlet approach.
Edited by L R


arova The extracts were assessed for their anti-oxidant activity using DPPH, ABTS and nitric oxide radical inhibition
Keywords:
assays and anti-arthritic activity through membrane stabilization assay, anti-proteinase activity and protein
Zingiber officinale denaturation inhibition assays. The highest scavenging potential was exhibited by Z. officinale methanolic
Antioxidant extract (ZOME) as assessed by DPPH assay (86.26 § 0.97%), ABTS assay (91.04 § 0.96%) and nitric oxide assay
Anti rheumatic (86.72 § 1.51%). Similarly, the ZOME showed 84.72 § 1.38% of inhibition in membrane stabilization assay,
8-Gingerol 82.72 § 1.48% in anti-proteinase activity and 81.68 § 1.66% in protein denaturation inhibition capacity. GC-
Bioavailability MS analysis of ZOME showed 30 different phytoconstituents. The major ginger bioactive compounds were
In silico virtually docked with novel RA targets using PyRx and the pharmacokinetic properties like ADMET proper-
ties were evaluated. Among all, 8-Gingerol showed the highest covalent interaction along with suitable bind-
ing affinity to the RA targets of COX-II (
7.2), TNF-a (
4.7), MCSF (
5.0), IL-1b (
5.7) and MMP9 (
6.7) kcal/
mol. 8-Gingerol can be a better drug candidate that must be further investigated for mechanistic studies to
verify its therapeutic potential in treating RA.
© 2019 Published by Elsevier B.V. on behalf of SAAB.

1. Introduction
Rheumatoid Arthritis (RA) is a chronic, autoimmune disorder pri-
marily affecting the synovial joints and damages the cartilage and
bones. RA annually affects about 0.5
1% of population in worldwide
and women are mostly affected than men at 3:1 ratio (Guo et al.,
2018; Silman and Pearson, 2002). RA is strongly associated to the auto-
immune response triggered by various genetic, epigenetic and some
environmental factors and its progression is classified into three stages
as swelling with pain, inflamed synovium, finally bone and cartilage
destruction causing joint damage with pain (Smolen et al., 2018). Vari-
ous proinflammatory cytokines (Interleukin-1b, 17, 34, TNF-a:
Tumour Necrosis Factor), enzymes of Cyclooxygenase II (COX-II), Lysyl
Oxidase (LOX), Prostaglandin-Endoperoxide Synthase (PTGS), Reactive
Oxygen Species (ROS) of NO, H2O2, prostaglandins, Macrophage Colony
Stimulating Factor (MCSF), Transforming Growth Factor (TGF) are
mainly involved in the RA pathology. Currently PTGS, COX and LOX
* Corresponding author.
E-mail address: drsureshbiotech2003@gmail.com (S. Periyasamy).
are the most potential target proteins for RA treatment at chronic
inflammatory conditions. The chemodrugs used for the treatment of
arthritis includes steroidal drugs (corticosteroid), Non-Steroidal Anti-
Inflammatory Drugs (NSAIDs) (Ibuprofen and naproxen), Disease-
Modifying Anti-Rheumatic Drugs (DMARDs) (Methotrexate, lefluno-
mide). Even biological therapies such as infliximab (TNF-a blocking
agents), rituximab (anti-CD 20 therapy) and abatacept are prescribed
for RA treatment (Joshi and Dhaneshwar, 2014). Chemo and biological
therapies suppresses the inflammation and provide only temporary
relief along with adverse side effects. Administration of Classic
DMARDs like methotrexate are reported with side effects like nausea,
vomiting, kidney malfunctioning, haematological adverse state includ-
ing anemia and thrombocytopenia (Gowdie, 2011; Kumar et al., 2016).
Herbal or natural medicines are an alternative treatment to the
harmful disease with lesser side effects (Murugesan et al., 2019;
Rengasamy et al., 2019). In India, there are more than 2500 plants
species that are currently used as herbal medicaments (Pan et al.,
2013; Rengasamy et al., 2019). Further, many of the crude herbal-
based drugs and their constituents have a potent antioxidant activity
and scavenge free radicals, since these free radicals propagate

https://doi.org/10.1016/j.sajb.2019.12.019
0254-6299/© 2019 Published by Elsevier B.V. on behalf of SAAB.
46 S. Murugesan et al. / South African Journal of Botany 130 (2020) 45
53
inflammatory responses and cause cartilage damage (Suroowan and
Mahomoodally, 2018). Natural chemical constituents extracted from
medicinal plants can effectively interact and modulate the expression
of pro-inflammatory signal on inflammation pathway, and reduces
the adverse arthritic effect (Amran et al., 2015).
Zingiber officinale Rosc. (ginger) is a rhizomatous, monocotyledon-
ous plant that is extensively used around the world to treat a wide
range of diseases such as rheumatic disorders, cold symptoms, fevers,
gastrointestinal complications, motion sickness, bronchitis, and dia-
betes (Brahmbhatt et al., 2013). The biological and medicinal proper-
ties of Z. officinale were mainly attributed through its bioactive
constituents like 6-Gingerol, 6-shogaol, other phenolics and flavo-
noids. Though NSAIDs and DMARDs are prescribed during chronic
inflammation conditions, their long term usage ends up with gastro
intestinal disorders. Ginger being another potent source with rich
secondary metabolites was proven to suppress the prostaglandin
synthesis by inhibiting COX-I, COX-II, suppressing the leukotriene
biosynthesis through 5-LOX inhibition and inhibiting the several
inflammatory cytokines expressions (Grzanna et al., 2005). In fact,
the prime compound, 6-Gingerol was reported with various pharma-
cological activities including anti-inflammatory, antioxidant, antican-
cer, anti-lipidemic, antidiabetic, analgesic and antipyretic properties
(Mahomoodally et al., 2019; Srinivasan, 2017). which can be utilized
in treating the arthritis and its associated inflammatory responses.
Considering the potential of ginger metabolites, the present study
was attempted to investigate the anti-rheumatic properties of Z. offi-
cinale using in silico and in vitro approaches.
2. Materials and methods
2.1. Sample collection, authentication and extraction
The fresh rhizomes of ginger (Zingiber officinale Rosc.) were col-
lected from Ooty, Tamil Nadu, India and authenticated by Botanical
Survey of India (BSI) Coimbatore, Tamil Nadu, India. (BSI/ SRC/ 5/ 23/
2018/ TECH/ 2242). Shadow dried ginger was grided into powder and
used for the extraction (10 g of ginger powder in 150 mL of different
organic solvents). Using soxhlet apparatus the phytoconstituents
were extracted with different organic solvents (Methanol, Ethyl ace-
tate, Hexane). Extracts were concentrated through rotary vacuum
evaporator at 45 °C for 10 mins (Yamato Scientific Co, Japan) The final
concentrated extracts were stored in
20 °C and used for In vitro
assays.
2.2. In vitro scavenging potential assays
Antioxidant potential of different extracts of Z. officinale was
determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,20 -Azino-
bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and Nitric oxide
(NO) activities and compared with standard butylated hydroxyto-
luene (BHT)
2.3. DPPH free radical scavenging activity
Different concentration of ginger extracts (20, 40, 60, 80 and
100 mg/mL) were added to 3 mL of DPPH solution (0.135 mM). After
20 min of incubation, the absorbance (Abs) was measured at 517 nm
and compared with the control. DPPH radical scavenging activity was
indicated by its decreasing absorbance at 517 nm with respective
concentrations (Jeyadevi et al., 2019).
ðAbs con
Abs sampleÞ
% Inhibition activity ¼  100 ð2:1Þ
ðAbs conÞ
Where, Abs con is the absorbance of the control and Abs sample is
the absorbance of the sample extract.
2.4. ABTS radical cation decolorization assay
Free radical scavenging activity of Z. officinale was determined by
ABTS radical cation decolorization assay. Various concentrations of
different ginger extract (20, 40, 60, 80 and 100 mg/mL) was added to
3 mL of ABTS¢+ cation radical and incubated at room temperature for
30 min. Then, the sample was read at 734 nm and the percentage of
inhibition was calculated using Eq. (2.1) (Jeyadevi et al., 2019).
2.5. Nitric oxide radical scavenging assay
Aqueous solution of sodium nitroprusside spontaneously gener-
ates NO at physiological pH, which interacts with oxygen to produce
nitric oxide that can be measured through calorimetrical method.
3 mL of reaction mixture containing sodium nitroprusside (10 mM in
PBS: Phosphate buffer saline) and 1 mL of plant extracts were incu-
bated at 25 °C for 3 h. After incubation, 0.5 mL of reaction mixture
was removed and 0.5 mL of Griess reagent (1% sulfanilamide, 5%
H3PO4, 0.1% naphthalene diamine dihydrochloride) was added. The
absorbance was read at 546 nm (Atere et al., 2018).
2.6. In vitro anti-arthritic activities
Z. officinale extracts obtained from various organic solvents were
tested for the anti arthritic activities and results were compared with
standard anti-inflammatory drug (diclofenac).
2.7. Protein denaturation inhibition assay
Bovine serum albumin (5% of 2.4 mL) and 1 mL aqueous solution
contains crude plant extracts were added in the reaction tube. The pH
was adjusted to 6.3 and incubated at 37 °C for 20 min. Then the sam-
ples were heated at 57 °C for 30 min. After cooling the samples, 2.5 mL
PBS (pH 6.3) was added to each tube and made up to 5 mL. Turbidity
was determined in spectrophotometer at 660 nm wavelength com-
pared to control. The percentage inhibition of protein denaturation
was computed using Eq. (2.1) (Senthilkumar et al., 2017).
2.8. Membrane stabilization assay
To the assay reaction mixtures (4.5 mL) containing 1 mL of 0.15 M
PBS (pH 7.4), 2 mL hypotonic saline solution (0.25% NaCl), and 1 mL
aqueous solution containing Z. officinale extract, 0.5 mL human red
blood cell (HRBC) suspension [10% v/v] in normal saline was added.
For control, 1 mL of hypotonic saline was used instead of test solution
while red blood cells were absent in the control. The mixture was
incubated at 57 °C for 30 min. The tubes were chilled and then centri-
fugation was carried out, finally absorbance of the supernatants was
determined at 560 nm. Percentage of membrane stabilizing potential
was calculated by Eq. (2.1) (Sharma et al., 2016).
2.9. Proteinase inhibitory assay
The reaction mixtures of 2 mL contained 0.06 mg trypsin, 1 mL of
25 mM tris-HCl buffers (pH 7.4) and 1 mL of Z. officinale extract. The
mixtures were incubated at 37 °C for 10 min, and then 1 mL of 0.8%
of casein solution was added with 20 min incubation. Later, 2 mL of
70% of perchloric acid was added to stop the reaction proceeding. The
cloudy suspension was centrifuged and supernatant solution was
measured at 280 nm wavelength against buffer as blank. The percent-
age of inhibition was calculated as per the Eq. (2.1) (Aziz, 2015).
2.10. Gas Chromatography Mass Spectrometry (GC-MS) analysis
The active phytochemicals responsible for the antioxidant and
anti inflammatory activity in the crude extract of Z. officinale was
 S. Murugesan et al. / South African Journal of Botany 130 (2020) 45
53 47

examined using gas chromatography mass spectrometry (GC 7890-
MS 5975C) Electron ionization mode with Triple-Axis Detector (Agi-
lent Technologies, Santa Clara, CA, USA). The capillary column of
DB5MS with internal diameter: 30 m £ 0.25 mm, film thickness:
0.25 mm was used for separation process. The sample injector tem-
perature was 250 °C in splitless mode and helium was used as a car-
rier gar with the flow rate of 1 mL/min. The MS spectral data was
obtained from the Compound Library database of NIST08s.LIB
(National Institute of Standards and Technology, US) based on the
retention time and mass.
2.11. Computational studies
bioavailability and mutagenicity were estimated using admetSAR
server (Yang et al., 2019).
2.14. Preparation of target RA proteins
Crystalline structure of chief targets like COX-II (5F19), TNF-a
(1THF), MCSF (5LXF), IL-1b (9ILB), MMP9 (1L6J) proteins were
retrieved from RCBS-PDB (http://www.rcsb.org/pdb). Water mole-
cules, ligands and undesired bonding were removed from crystalline
structure of target proteins using Discovery Studio 2017R2. The miss-
ing hydrogen atoms, unfilled atom valency were ratified and energy
minimization was achieved by force-field (Kumar et al., 2020).

The major GC-MS peak of bioactives was selected for further in silico
analysis. The chief RA target proteins were selected from DrugBank
Database and Literature reports. Molecular docking analysis was per-
formed on AutoDock (AD) inbuild vina PyRx 0.8 version software and
the interaction studies were carried out on Discovery Studio 2017.
2.12. Preparation of ligands and analysis of drug likeliness
The crystal 3D structure of selected active compounds in metha-
nolic extract of Z. officinale was obtained from PubChem Database.
Drug-likeliness properties including Topological polar surface area
(TPSA), Atom Molar Refractivity (AMR), log P, Alog P, and physico-
chemical properties were analysed for the selected active compounds
using DruLiTo software (Lipinski et al., 2001).
2.15. Molecular docking studies
Initially, the energy minimization process was performed to the 3D
structures of selected ligands using MMFF94 force field. The energy
minimized ligands (.sdf) and protein structures (.pdb) were converted
into PDBQT file format (.pdbqt). The protein-ligand docking studies
were achieved by grid box approach for covering the binding sites. Eval-
uation of docking results was sorted via predicted binding energy
(-kcal/mol). A cluster analysis based on root mean square deviation val-
ues (RMSD values) and the lowest energy conformation of the most
populated cluster was considered as the most trustable solution (Aru-
mugam et al., 2019).
2.16. Binding poses visualization and analysis of interactions

2.13. Pharmacokinetic and bioactivity properties
The pharmacokinetic properties like human intestinal absorption
(HIA), water solubility, distribution (blood-brain barrier (BBB) perme-
ability log BS, central nervous system (CNS) permeability), metabo-
lism (Cytochrome P450 inhibitors), excretion (renal and total
clearance), and toxicity (hepatotoxicity, carcinogenicity) were evalu-
ated using the pkCSM web server (Pires et al., 2015). The oral
The PyRx results were analysed using Discovery Studio 2017R2.
The different binding mode of ligands file obtained from the end of
the docking studies and that files were analysed on Discovery Studio
to check the best binding mode with the binding site cavity. The
protein-ligand interactions of H-bond, C

H bond, Vander Waals
interaction, Alkyl, Pi-Alkyl, and Pi-Pi Alkyl interactions were viewed
and various atomic distance, angle were measured the same software
(Arumugam et al., 2019; Kumar et al., 2020).

Fig. 1. In vitro Antioxidant and Anti arthritic activities of ZOME. (a) ABTS radical scavenging activity of different crude extracts of Zingiber officinale. (b) DPPH radical scavenging
activity of different extracts of Zingiber officinale. (c) NO radical scavenging activity of different extracts of Zingiber officinale. (d). Protein denaturation inhibition capacity of different
extracts of Zingiber officinale. (e) Anti proteinase effect of different extracts of Zingiber officinale. (f) Membrane stabilization properties of different extracts of Zingiber officinale.
48 S. Murugesan et al. / South African Journal of Botany 130 (2020) 45
53
2.17. Statistical analysis
The in vitro assay results were calculated as means § SD (n = 3).
Data were analysed by comparing the different ginger extracts with
appropriate standard.
3. Results
3.1. In vitro studies
ROS formation increases the oxidative stress that leads to autoim-
mune diseases including RA. The increasing lipid peroxidation
reduces the anti oxidant enzymes activities and decreases the Na⁺/
K⁺-ATPase functions. Increasing ROS level also damage the immuno-
globulins, and affect the mechanism of DNA mismatch repair. From
these previous reports, the ROS oxidative stress is an important path-
ological role in RA progression (Di Dalmazi et al., 2016; Staron
et al.,
2012). In our investigation, the potential antioxidant activities of
crude extract of Z. officinale was evaluated by DPPH, ABTS and NO
assays, and the inhibitory effect was compared with the standard
BHT. Among these extracts, methanolic extract showed the signifi-
cant activity in a dose dependent manner. Highest antiradical poten-
tial was obtained from the ZOME at the maximum tested
concentration (100 mg/mL). The percentage inhibition of ZOME was
found to be 86.26§0.97% in DPPH assay which was little lower when
compared to the standard BHT activity (94.52§1.63%) and exhibited
higher activity than the other extracts (Fig 1. a). Regarding the ABTS,
Fig. 2. The GCMS chromatogram of the methanolic extract of Zingiber officinale Rosc.
ZOME exhibited 91.04§0.96% of inhibitory activity and least activity
exhibited by ZOHX (57.43§1.81%) (Fig 1. b). ZOME also showed the
highest NO scavenging potential of 86.72§1.51% that was compara-
ble with the activity of BHT (97.35§0.88%) (Fig 1. c).
Protein denaturation mechanism plays a crucial role in inflamma-
tory pathway by increasing the auto antigen production in RA pathol-
ogy (Ludwig et al., 2017). NSAIDs like provides protective effect
against denaturation of protein (Saso et al., 2001). With respect to
the protein denaturation inhibitory assay using different ginger
extracts, ZOME showed significant inhibition with the higher inhibi-
tion capacity (81.68§1.66%) (Fig 1. d).
Proteinase has been closely related to the arthritic mechanism
since it degrades the matrix tissues during the inflammation. Espe-
cially, MMPs are endopeptidases enzymes mainly involved in the car-
tilage matrix degradation and cause the destruction of bone joints
(Itoh, 2017). The crude extract of Z. officinale contributed the anti-
proteinase activity, yet again the ZOME showed higher proteinase
inhibitory effect (82.72§1.48%) compared with Standard diclofenac
(90.35§1.38%). In this particular assay, the inhibitory effect of the
standard was higher when compared to the ZOME (Fig 1. e).
Lysis of lysosomes during inflammation releases harmful enzymes
in to the circulatory system. NSAIDs are used to suppress the release
of lysosomal enzymes by neutralizing the membrane damage of lyso-
somes. Undesirable environmental conditions like hypotonic
medium, over heat, and chemical medications containing Methotrex-
ate, hydroxychloroquine may affect the stability of RBC membrane
and causes hemolysis (Held et al., 2018). Since RBCs mimics the
 S. Murugesan et al. / South African Journal of Botany 130 (2020) 45
53 49

Table 1 Table 3
Bioactive compounds derived from Zingiber officinale methanol Molecular docking of selected GCMS compounds with RA target proteins.
extract.
Compounds COX-2 IL1-Beta MCSF MMP-9 TNF Alpha
S. no RT % Peak Compound name
2,5-dibutylfuran
6.5
4.3
4.7
5.7
4.3
1 9.709 0.76 Decanal 6-Gingerol
7.8
5.7
5
6.7
4.7
2 13.417 7.76 Zingiberene 8-Gingerol
7.5
5.4
5.7
6.1
5.6
3 13.467 5.39 Curcumene Benzoic acid
6.1
4.6
4.9
6.2
4.8
4 13.534 2.47 Beta-bisabolene Dihydrocapsaicin
7.4
5.3
5.4
5.9
5.5
5 13.778 1.13 Germacrene D Dihydropseudoionone
4.4
3.2
3.6
4.9
3.4
6 13.861 5.02 Beta-sesquiphellandrene Ferulic acid ethyl ester
7.3
5.3
5.1
7.1
5.4
7 14.373 0.73 Nerolidol Geranylacetone
4.4
3.4
3.7
5
3.3
8 15.22 0.57 Alpha-terpineol Zingerone
6.9
5.2
5
7.4
5
9 15.506 0.54 Terpineol
10 16.017 0.55 Gamma-elemene
11 16.21 0.53 Beta-eudesmol secondary metabolites were identified (Fig 2). The polyphenol of 6-
12 16.252 0.85 Alpha-cedrene Gingerol the major peak (23.46%) on 23.25 min, followed by hexade-
13 16.537 2.07 Dihydropseudoionone
14 17.099 6.27 Zingerone
canoic acid (10.04%), zingiberene (7.76%), dihydrocapsaicin (7.37%),
15 17.242 0.61 Curcuphenol zingerone (6.27%), curcumene (5.39%), beta bisabolene (5.02%) octa-
16 18.005 2.32 Farnesol, acetate decanoic acid (4.78%) and 8-Gingerol (4.45%) (Table 1). The active
17 18.819 1.63 Geranylacetone compound, 6-Gingerol was reported for many potential biological
18 20.648 0.93 9,12-Octadecadienoic acid
properties including anti-cancer, antioxidant, anti-inflammatory and
19 21.386 1.32 Benzoic acid
20 22.519 0.81 3-Acetamidobenzoic acid inhibit the angiogenesis activities (Wang et al., 2014).
21 22.586 1.39 2,5-dibutylfuran
22 23.257 23.46 6-Gingerol
23 23.475 4.03 Ferulic acid ethyl ester
3.2. In silico studies
24 23.701 0.54 Carinol
25 24.255 10.22 Hexadecanoic acid
26 24.876 4.45 8-Gingerol
3.2.1. Drug-likeness properties
27 25.044 0.65 1,4-naphthoquinon The physiochemical properties of major 16 active compounds
28 25.798 4.78 Octadecanoic acid were analysed on DruLiTo. Around nine compounds obeyed the Lip-
29 26.428 7.37 Dihydrocapsaicin inski’s rule, especially, 6-Gingerol and 8-Gingerol showed higher
30 26.57 0.85 P-Toluic acid, pentadecyl ester
TPSA (66.76, 66.76) and AMR (80.8, 86.63) with suitable HBA, HBD
(Table 2) and nRB. TPSA and AMR are important physiochemical
lysosomal membrane, studying the protective effect of could be use- properties mainly involved in drug absorption, transport and pene-
ful to analyze the anti-inflammatory property. Here the crude extract tration mechanism (Ertl et al., 2000).
of ZOME expressed the greater stabilization effect (84.72§1.38%)
when compared to other extracts and also found to exhibit equiva- 3.2.2. Molecular docking studies
lent activity when compared with the BHT (93.35§1.33%) (Fig 1. f). The selected bioactives were docked with RA target proteins (COX-II,
Various biological studies have shown that many phenolic and fla- IL-1b, MCSF, MMP9 and TNF-a) using PyRx. Among these compounds,
vonoids significantly contributed to the antioxidant and anti-arthritic dihydrocapsaicin, 6-Gingerol and 8-Gingerol expressed the minimized
activities in herbal plants. Z. officinale, has abundant amount of poly- binding energy in kcal/mol (Table 3). COX-II with 6-Gingerol, and IL-1b
phenols like gingerol derivatives, shogaol, paradol and zingerone with 6-Gingerol showed the highest binding affinity (
7.8, and
which might be responsible for the anti arthritic activities (Funk
5.7 kcal/mol). Similarly, growth factor molecule of MCSF with
et al., 2009; Semwal et al., 2015). 8-Gingerol and signaling molecule of TNF-a with 8-Gingerol exposed the
Since ZOME exhibited best antiradical and antiarthritic activities, potential binding affinity with minimized binding energy (
5.7 and
it was further chosen for GC
MS analysis, because it exhibited excel-
5.6 kcal/mol). Zingerone with endopeptidase of MMP9 complex exhib-
lent antiradical activities. Using GC
MS results, 30 different ited the higher binding score with lesser binding energy (
7.4 kcal/mol).

Table 2
Physicochemical properties of the active compounds and accordance with the rules of drug-likeness.

Ligand Compounds MW log P Alog P HBA HBD TPSA AMR nRB

1 2,5-Dibutylfuran 180.15 3.963
0.65 1 0 9.23 53.26 6

2 Hexadecanoic acid 256.24 7.57
3.898 2 1 37.3 55.52 14
3 6-Gingerol 294.18 2.437
1.597 4 2 66.76 80.8 10
4 8-Gingerol 322.21 3.575
2.173 4 2 66.76 86.63 12
5 Benzoic acid 122.04 0.982 0.968 2 1 37.3 36.96 1
6 Beta-Sesquiphellandrene 204.19 6.399 3.192 0 0 0 68.91 4
7 Bisabolene 204.19 5.551 3.375 0 0 0 69.93 4
8 Curcumene 202.17 5.761 3.855 0 0 0 71.56 4
9 Dihydrocapsaicin 307.21 4.503
0.766 4 2 58.56 85.05 11
10 Dihydropseudoionone 194.17 3.618 2.825 1 0 17.07 63.79 6
11 Farnesol acetate 264.21 5.086 3.951 2 0 26.3 84.37 9
12 Ferulic acid ethyl ester 222.09 1.524 0.769 4 1 55.76 64.51 5
13 Geranylacetone 194.17 3.619 2.825 1 0 17.07 63.79 6
14 Germacrene D 204.19 6.294 2.797 0 0 0 69.55 1
15 Zingerone 194.09 0.761
0.011 3 1 46.53 58.17 4
16 Zingiberene 204.19 6.354 3.339 0 0 0 70.13 4
50 S. Murugesan et al. / South African Journal of Botany 130 (2020) 45
53

Table 4
Interacting residues of RA targets with selected bioactive compounds from Z. officinale.

Compounds COX-II IL-1b MCSF MMP9 TNF-a

6-Gingerol TRP387, HIS388, LEU391, ASN7, GLU64, LEU62, LYS65, ARG66, PHE67, MET65, ARG51, THR96, LEU39, ILE154, SER9, ILE155, VAL13
HIS386, TYR385 SER153, LYS63 PRO110, LEU113, LEU114 LEU44
8-Gingerol ASN382, HIS386, HIS207, SER125, LEU134, ASP142, GLN58, ASP59, LEU55, LYS88 ARG51, TRY52, ASN38, TYR59, PRO8, ILE154, THR7,
VAL295, ILE408, LEU391, PRO131, PHE133 ASP182, LYS184, ARG95, VAL13, LEU36, ILE155
TYR404 LEU39
Zingerone ALA199, HIS386, ALA202, SER43, LEU62, PRO91 TYR49, LYS116 HIS401, LEU397, LEU418 ARG6, LEU36, PRO8, VAL13
GLN203

3.2.3. Interaction studies important properties for oral drug designing process. All the com-
The rigid docking results were visualized using Discovery studio pounds showed above 90% of intestinal absorption value with suit-
for analysis of interaction. The best binding poses of protein-small able aqueous solubility (8-Gingerol: 91.716; 6-Gingerol: 92.416, and
molecule interactions were visualized and tabulated in Table 4. The zingerone: 94.103). The in silico analysis of compound distribution
strongest interaction was observed in the 8-Gingerol with RA targets (log BB; log PS), metabolism (cytochrome P450 inhibitors) excretion
protein complexes. COX-II with 8-Gingerol complex formed one H- (total clearance and renal clearance) and toxicity (oral toxicity, hepa-
 
bond (ASN382; 1.95 A), two Pi-Pi stacked interaction (HIS386; 4.18 A, totoxicity, AMES toxicity) were analysed and tabulated in Table 5. All
 
HIS207; 4.34 A), three alkyl interaction (VAL295; 4.61 A, ILE408; the compounds did not show any toxicity including AMES, oral acute
  
4.79 A, LEU391; 5.43 A) and one Pi-Alkyl interaction (TYR404; 5.13 A) and hepatotoxicity. Carcinogenicity and mutagenicity have not been
(Fig 3). The protein-small molecule complex of 8-Gingerol-MCSF observed in any compounds from the in silico prediction. Overall 8-

showed one hydrogen bonding (GLN58; 3.38 A), one C

H bonding Gingerol showed higher oral availability as predicted by admetSAR.

(ASP59; 3.3 A) and two vander waals attraction (LEU55, LYS88). The The dosage of maximum tolerance was predicted for all three com-
complex of 8-Gingerol and MMP9 exhibited the three covalent bonds pounds, 8-Gingerol (0.694), 6-Gingerol (0.635), and zingerone
  
(ARG51; 3.20 A, TRY52; 2.89 A, ASN38; 2.72 A), two CH bond (0.544). Using Molinspiration Cheminformatics web server, all
 
(ASP182; 3.59 A, LYS184; 3.52 A) and two hydrophobic interactions selected compounds relative bioactivity scores were predicted and
 
(ARG95; 3.76 A, LEU39; 4.69 A). The receptor-ligand complex of TNF- summarized in Table 5. The highest score indicates better biological
a with 8-Gingerol

significantly interacted with three covalent bonds
 
activities. In an over view, 8-Gingerol and 6-Gingerol showed better
(TYR59; 2.89 A, PRO8; 2.99 A, ILE154; 2.11 A), one carbon hydrogen bioactivity score and acts as an enzyme inhibitor (0.34 and 0.38).
 
bond (THR7; 3.36 A), one Pi-Sigma interaction (VAL13; 3.92 A) and
 
two Pi-Alkyl interaction (LEU36; 5.06 A, ILE155; 4.98 A). 8-Gingerol
 4. Discussion
with IL-1b complex formed the three covalent bond (SER125; 2.88 A,
 
LEU134; 2.40 A, ASP142; 2.A), one C

H bond (PHE133) and three
RA being a chronic autoimmune disorder is manifested with red-
hydrophobic interactions (PRO131, PHE133).
dish joints, swelling, and fatigue along with increased mortality rate.
6-Gingerol with RA target protein complexes also shown notable
Initially, the synovial fluid lining the joints is majorly affected fol-
strongest interaction. Especially, protein-ligand complex of 6-Gingerol
 lowed by cartilage and bone damages which may even worsen up to
with IL-1b formed the 5 H-bond interactions (ASN7; 1.98 A , GLU64;
    multi organ failure. Women are at the higher risk end with the arthri-
2.79 A, LEU62; 2.63 A, LYS65; 2.07 A, SER153; 1.96 A) and one Pi-Alkyl
 tis incidence along with other striking causes including genetic fac-
interaction (LYS63; 4.75 A) (Fig 3). The polyphenolic compound Zinger-
tors, stress, smoking and viral infections (Mcinnes and Schett, 2011)
one had shown significant interaction to the RA targets with suitable
The aforementioned factors influence the post transcriptional alter-
covalent and hydrophobic interactions shown in Table 4.
ations and it releases the citrullinated proteins, rheumatoid factors and
other self-proteins like vimentin, a-enolase, histones, fibronectin,
3.2.4. Pharmacokinetic and bioactivity properties fibrinogen, and collagen (Kurowska et al., 2017). These proteins are
From the docking and visualization studies, 8-Gingerol, 6- not considered as self proteins, so Anti citrullinated protein antibodies
Gingerol and Zingerone were selected for pharmacokinetic and bio- (ACPAs) are produced in response to it. Several inter and intra cellular
activity analysis. The water solubility and intestinal absorption are signaling mechanisms are involved in the formation of immune

Fig. 3. Schematic diagrams of RA targets with 8-gingerol complex interaction using Discovery studio. (a) IL-1b with 8-Gingerol. (b) MMP9 with 8-Gingerol.
 S. Murugesan et al. / South African Journal of Botany 130 (2020) 45
53 51

Table 5
Pharmacokinetic and bioactivity properties of selected active compounds from Z. officinale.

ADMET Properties name Compound names

Zingerone 6-Gingerol 8-Gingerol

Bioavailability Human oral bioavailability 0.7857 0.7714 0.8000

Absorption Water solubility (log S mol/L)
1.7
3.164
3.795
Intestinal absorption (%) 94.103 92.416 91.716
Distribution BBB permeability (log BB) 0.006
0.727
0.794
CNS permeability (log PS)
2.175
2.788
2.799
Metabolism CYP2D6 substrate (Yes/No) No No No
CYP3A4 substrate (Yes/No) No No Yes
Excretion Total Clearance (log ml/min/kg) 0.307 1.339 1.4
Renal OCT2 substrate (Yes/No) No No No
Toxicity AMES toxicity (Yes/No) No No No
Max. tolerated dose human (mg/kg/day) 0.544 0.635 0.694
Oral Rat Acute Toxicity LD50 (mol/kg) 2.129 1.958 1.957
Hepatotoxicity (Yes/No) Yes No No
Carcinogenicity (Yes/No) No No No
Mutagenicity (Yes/No) No No No
Bioactivity score GPCR ligand
0.58 0.16 0.18
Ion channel modulator
0.18 0.04 0.03
Kinase inhibitor
1.15
0.33
0.27
Nuclear receptor ligand
0.59 0.2 0.23
Protease inhibitor
0.72 0.15 0.2
Enzyme inhibitor
0.07 0.38 0.34

Fig. 4. Rheumatoid arthritis progression mechanism and pathology: RANKL- Receptor activator of nuclear factor kappa-B ligand; TGF-b- Transforming growth factor beta; MMPs-
Matrix metalloproteinases; M-CSF- Macrophage Colony-Stimulating Factor; GM-CSF- Granulocyte Monocyte Colony-Stimulating Factor COX-II- Cyclooxygenase-2; IL17- Interleukin
17; IL34- Interleukin 34; Th1- T helper type 1, Th17- T helper type 17.

complex. Mast cells, macrophages and natural killer cells are the vital
players in the pathology of RA progression. TNF-a, IL-1, IL-6, IL-12, IL-
15, IL-18, and IL-23 are few cytokines secreted by macrophages, espe-
cially TNF-a and IL-6 regulates the cytokines and chemokines activa-
tion that aggravate inflammatory reaction and causes cartilage
damage (Smolen et al., 2018) (Fig 4). Nowadays, extra cellular specific
biological DMARDs like sarilumab and adalimumab are available to
reduce the inflammation. However, NSAIDs simply acts as timely pain
subsidizers rather having a long term mechanistic modification in pre-
venting the joint and bone damage (Rein and Mueller, 2017).
Nearly 60% of people look for an alternative herbal remedy for the
long term cure of RA (Rao et al., 1999) Chopra et al., 2000 demon-
strated the antiarthritic potential of an ayurvedic tablet, RA-1 contain-
ing of Boswellia serrata, Withania somnifera, Z. officinale and Curcuma
longa in 182 patients for 4 months. From the increase in the daily dose
of 444 mg, almost 50% of arthritic patients showed significantly
reduced the swelling of bones when compared with the placebo
patients. Studies using herbal medicines still need improvised and
standardized trials considering the efficacy and safety for the global
recognition of traditional herbal medicines (Chopra et al., 2000).
52 S. Murugesan et al. / South African Journal of Botany 130 (2020) 45
53
Our investigation majorly focused on searching of nutraceutically
valuable new molecules from herbal plant of Z. officinale with suitable
biological efficacy and least toxicity for treatment of arthritis. The
present study has revealed that the antiradical and anti-arthritic
activity of methanolic extract of the Z. officinale through in vitro and
in silico studies. From this result, 8-Gingerol is specifically chosen for
the further study. 6-Gingerol, 8-Gingerol and 10-Gingerol are few
non-volatile, pungent, lipophilic compounds of ginger rhizome,
which are reported as DPPH, super oxide and hydroxyl radical scav-
enging agents (Baliga et al., 2015). Other active ingredients in ginger
including paradol, shogaol showed strong inhibition of COX-II
enzyme that is involved in inflammatory responses (Van Breemen
et al., 2011). No such proof is available to substantiate the anti
arthritic mechanistic role of 8-Gingerol. Owing to the increasing
demand for the gingerol content in the medical care, 8-Gingerol
content is being mass cultured using callus suspension technique
using different elicitors and precursors that may intervene with the
parent phenylpropanoid pathway involved in gingerol production
(El-Nabarawy et al., 2015).
5. Conclusion
The present study demonstrated that the methanolic extract of
Zingiber officinale favourably prevented the ROS damage and sup-
presses the inflammatory responses. GC-MS analysis revealed the
presence of 30 bioactives. The selected bioactive compounds upon
docking with arthritic targets revealed that 8-Gingerol, 6-Gingerol
and Zingerone as the best anti-arthritic compounds. With further
screening, 8-Gingerol was selected as the chief arthritic compound of
interest that is being produced in Zingiber officinale through the callus
culture techniques using elicitors to adopt a cost effective technique
in understanding the mechanism of action of 8-Gingerol in both the
cell line and animal models.
Declaration of competing interest
All authors declared that there are no conflicts of interest
Acknowledgments
We acknowledge Department of Science and Technology-Science
and Engineering Research Board (DST-SERB) New Delhi, India for pro-
viding the financial support to conduct this study (Project no.: EEQ/
2017/000447).
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